Structure-activity relationships for unit C pyridyl analogues of the tuberculosis drug bedaquiline

The ATP-synthase inhibitor bedaquiline is effective against drug-resistant tuberculosis but is extremely lipophilic (clogP 7.25) with a very long plasma half-life. Additionally, inhibition of potassium current through the cardiac hERG channel by bedaquiline, is associated with prolongation of the QT interval, necessitating cardiovascular monitoring. Analogues were prepared where the naphthalene C-unit was replaced with substituted pyridines to produce compounds with reduced lipophilicity, anticipating a reduction in half-life. While there was a direct correlation between in vitro inhibitory activity against M. tuberculosis (MIC₉₀) and compound lipophilicity, potency only fell off sharply below a clogP of about 4.0, providing a useful lower bound for analogue design. The bulk of the compounds remained potent inhibitors of the hERG potassium channel, with notable exceptions where IC₅₀ values were at least 5-fold higher than that of bedaquiline. Many of the compounds had desirably higher rates of clearance than bedaquiline, but this was associated with lower plasma exposures in mice, and similar or higher MICs resulted in lower AUC/MIC ratios than bedaquiline for most compounds. The two compounds with lower potency against hERG exhibited similar clearance to bedaquiline and excellent efficacy in vivo, suggesting further exploration of C-ring pyridyls is worthwhile.     

Variations in the C-unit of bedaquiline provides analogues with improved biology and pharmacology

Analogues of the anti-tuberculosis drug bedaquiline, bearing a 3,5-dimethoxy-4-pyridyl C-unit, retain high anti-bacterial potency yet exert less inhibition of the hERG potassium channel, in vitro, than the parent compound. Two of these analogues (TBAJ-587 and TBAJ-876) are now in preclinical development. The present study further explores structure-activity relationships across a range of related 3,5-disubstituted-4-pyridyl C-unit bedaquiline analogues of greatly varying lipophilicity (clogP from 8.16 to 1.89). This broader class shows similar properties to the 3,5-dimethoxy-4-pyridyl series, being substantially more potent in vitro and equally active in an in vivo (mouse) model than bedaquiline, while retaining a lower cardiovascular risk profile through greatly attenuated hERG inhibition.     

Block of the human ether-a-go-go-related gene (hERG) K channel by the antidepressant desipramine

Desipramine is a tricyclic antidepressant for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. Since blockade of cardiac human ether-a-go-go-related gene (hERG) channels is an important cause of acquired long QT syndrome, we investigated the acute effects of desipramine on hERG channels to determine the electrophysiological basis for its pro-arrhythmic potential. We examined the effects of desipramine on the hERG channels expressed in Xenopus oocytes using two-microelectrode voltage-clamp techniques. Desipramine-induced concentration-dependent decreases in the current amplitude at the end of the voltage steps and hERG tail currents. The IC₅₀ for desipramine needed to block the hERG current in Xenopus oocytes decreased progressively relative to the degree of depolarization. Desipramine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations, Tyr-652 located in the S6 domain of the hERG channel reduced the potency of the channel block by desipramine more than a mutation of Phe-656 in the same region. These results suggest that desipramine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of desipramine.     

Molecular determinants of loperamide and N-desmethyl loperamide binding in the hERG cardiac K+ channel

Abuse of the common anti-diarrheal loperamide is associated with QT interval prolongation as well as development of the potentially fatal arrhythmia torsades de pointes. The mechanism underlying this cardiotoxicity is high affinity inhibition of the human ether-a-go-go-related gene (hERG) cardiac K+ channel. N-Desmethyl loperamide is the major metabolite of loperamide and is a close structural relative of the parent molecule. To date no information is available regarding the affinity of N-desmethyl loperamide for human cardiac ion channels. The effects of N-desmethyl loperamide on various cloned human cardiac ion channels including hERG, KvLQT1/mink and Na_v1.5 were studied and compared to that of the parent. N-Desmethyl loperamide was a much weaker (7.5-fold) inhibitor of hERG compared to loperamide. However, given the higher plasma levels of the metabolite relative to the parent, it is likely that N-desmethyl loperamide can contribute, at least secondarily, to the cardiotoxicity observed with loperamide abuse. We used the recently solved cryo-EM structure of the hERG channel together with previously published inhibitors, to understand the basis of the interactions as well as the difference that a single methyl plays in the hERG channel blocking affinities of these two compounds.     

hERG1 channel activators: A new anti-arrhythmic principle

The cardiac action potential is the result of an orchestrated function of a number of different ion channels. Action potential repolarisation in humans relies on three potassium current components named I_Kr, I_Ks and I_K1 with party overlapping functions. The ion channel α-subunits conducting these currents are hERG1 (Kv11.1), KCNQ1 (Kv7.1) and Kir2.1. Loss-of-function in any of these currents can result in long QT syndrome. Long QT is a pro-arrhythmic disease with increased risk of developing lethal ventricular arrhythmias such as Torsade de Pointes and ventricular fibrillation. In addition to congenital long QT, acquired long QT can also constitute a safety risk. Especially unintended inhibition of the hERG1 channel constitutes a major concern in the development of new drugs. Based on this knowledge is has been speculated whether activation of the hERG1 channel could be anti-arrhythmic and thereby constitute a new principle in treatment of cardiac arrhythmogenic disorders. The first hERG1 channel agonist was reported in 2005 and a limited number of such compounds are now available. In the present text we review results obtained by hERG1 channel activation in a number of cardiac relevant settings from in vitro to in vivo. It is demonstrated how the principle of hERG1 channel activation under certain circumstances can constitute a new anti-arrhythmogenic principle. Finally, important conceptual differences between the short QT syndrome and the hERG1 channel activation, are evaluated.     

Improved BM212 MmpL3 Inhibitor Analogue Shows Efficacy in Acute Murine Model of Tuberculosis Infection

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED₉₉ of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery.     

hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656

The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is little mechanistic information on this action. We have investigated in detail hERG K(+) channel current (I(hERG)) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell I(hERG) measurements were made at 37 degrees C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I(hERG) tails at -40mV following a 2s depolarization to +20mV with an IC(50) value of 80nM. Comparable levels of I(hERG) inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I(hERG) was only weakly voltage-dependent, though the time dependence of I(hERG) inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I(hERG) inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K(+) channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore.     

Structural determinants for histamine H1 affinity,hERG affinity and QTc prolongation in a series of terfenadine analogs

In the late 1980’s reports linking the non-sedating antihistamines terfenadine and astemizole with torsades de pointes, a form of ventricular tachyarrhythmia that can degenerate into ventricular fibrillation and sudden death, appeared in the clinical literature. A substantial body of evidence demonstrates that the arrhythmogenic effect of these cardiotoxic antihistamines, as well as a number of structurally related compounds, results from prolongation of the QT interval due to suppression of specific delayed rectifier ventricular K+ currents via blockade of the hERG-IKr channel. In order to better understand the structural requirements for hERG and H₁ binding for terfenadine, a series of analogs of terfenadine has been prepared and studied in both in vitro and in vivo hERG and H₁ assays.     

Role of hERG potassium channel assays in drug development

Numerous structurally and functionally unrelated drugs block the hERG potassium channel. HERG channels are involved in cardiac action potential repolarization, and reduced function of hERG lengthens ventricular action potentials, prolongs the QT interval in an electrocardiogram, and increases the risk for potentially fatal ventricular arrhythmias. In order to reduce the risk of investing resources in a drug candidate that fails preclinical safety studies because of QT prolongation, it is important to screen compounds for activity on hERG channels early in the lead optimization process. A number of hERG assays are available, ranging from high throughput binding assays on stably expressed recombinant channels to very time consuming electrophysiological examinations in cardiac myocytes. Depending on the number of compounds to be tested, binding assays or functional assays measuring membrane potential or Rb+ flux, combined with electrophysiology on a few compounds, can be used to efficiently develop the structure-function relationship of hERG interactions.     

Role of hERG potassium channel assays in drug development

Numerous structurally and functionally unrelated drugs block the hERG potassium channel. HERG channels are involved in cardiac action potential repolarization, and reduced function of hERG lengthens ventricular action potentials, prolongs the QT interval in an electrocardiogram, and increases the risk for potentially fatal ventricular arrhythmias. In order to reduce the risk of investing resources in a drug candidate that fails preclinical safety studies because of QT prolongation, it is important to screen compounds for activity on hERG channels early in the lead optimization process. A number of hERG assays are available, ranging from high throughput binding assays on stably expressed recombinant channels to very time consuming electrophysiological examinations in cardiac myocytes. Depending on the number of compounds to be tested, binding assays or functional assays measuring membrane potential or Rb(+) flux, combined with electrophysiology on a few compounds, can be used to efficiently develop the structure-function relationship of hERG interactions.     

Evaluation of a high-throughput fluorescence assay method for HERG potassium channel inhibition

The number of projects in drug development that fail in late phases because of cardiac side effects such as QT prolongation can impede drug discovery and development of projects. The molecular target responsible for QT prolongation by a wide range of pharmaceutical agents is the myocardial hERG potassium channel. It is therefore desirable to screen for compound interactions with the hERG channel at an early stage of drug development. Here, the authors report a cell-based fluorescence assay using membrane potential-sensitive fluorescent dyes and stably transfected hERG channels from CHO cells. The assay allows semiautomated screening of compounds for hERG activity on 384-well plates and is sufficiently rapid for testing a large number of compounds. The assay is robust as indicated by a Z' factor larger than 0.6. The throughput is in the range of 10,000 data points per day, which is significantly higher than any other method presently available for hERG. The data obtained with the fluorescence assay were in qualitative agreement with those from patch-clamp electrophysiological analysis. There were no false-positive hits, and the rate of false-negative compounds is currently 12% but might be further reduced by testing compounds at higher concentration. Quantitative differences between fluorescence and electrophysiological methods may be due to the use- or voltage-dependent activity of the antagonists.     

Human ether-a-go-go related gene (hERG) K channels: Function and dysfunction

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in regulating cardiac excitability and maintenance of normal cardiac rhythm. Mutations in hERG cause a third of all cases of congenital long QT syndrome, a disorder of cardiac repolarisation characterised by prolongation of the QT interval on the surface electrocardiogram, abnormal T waves, and a risk of sudden cardiac death due to ventricular arrhythmias. Additionally, the hERG channel protein is the molecular target for almost all drugs that cause the acquired form of long QT syndrome. Advances in understanding the structural basis of hERG gating, its traffic to the cell surface, and the molecular architecture involved in drug-block of hERG, are providing the foundation for rational treatment and prevention of hERG associated long QT syndrome. This review summarises the current knowledge of hERG function and dysfunction, and the areas of ongoing research.     

On-chip constructive cell-network study (II): on-chip quasi-in vivo cardiac toxicity assay for ventricular tachycardia/fibrillation measurement using ring-shaped closed circuit microelectrode with lined-up cardiomyocyte cell network

Backgrounds

Conventional in vitro approach using human ether-a-go-go related gene (hERG) assay has been considered worldwide as the first screening assay for cardiac repolarization safety. However, it does not always oredict the potential QT prolongation risk or pro-arrhythmic risk correctly. For adaptable preclinical strategiesto evaluate global cardiac safety, an on-chip quasi-in vivo cardiac toxicity assay for lethal arrhythmia (ventricular tachyarrhythmia) measurement using ring-shaped closed circuit microelectrode chip has been developed.

Results

The ventricular electrocardiogram (ECG)-like field potential data, which includes both the repolarization and the conductance abnormality, was acquired from the self-convolutied extracellular field potentials (FPs) of a lined-up cardiomyocyte network on a circle-shaped microelectrode in an agarose microchamber. When Astemisol applied to the closed-loop cardiomyocyte network, self-convoluted FP profile of normal beating changed into an early afterdepolarization (EAD) like waveform, and then showed ventricular tachyarrhythmias and ventricular fibrilations (VT/Vf). QT-prolongation-like self-convoluted FP duration prolongation and its fluctuation increase was also observed according to the increase of Astemizole concentration.

Conclusions

The results indicate that the convoluted FPs of the quasi-in vivo cell network assay includes both of the repolarization data and the conductance abnormality of cardiomyocyte networks has the strong potential to prediction lethal arrhythmia.     

Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] Analogues with Bactericidal Efficacy against Mycobacterium tuberculosis Targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] (Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.     

A place for high-throughput electrophysiology in cardiac safety: screening hERG cell lines and novel compounds with the ion works HTTM system

Several commercially available pharmaceutical compounds have been shown to block the IKr current of the cardiac action potential. This effect can cause a prolongation of the electrocardiogram QT interval and a delay in ventricular repolarization. The Food and Drug Administration recommends that all new potential drug candidates be assessed for IKr block to avoid a potentially lethal cardiac arrhythmia known as torsades de pointes. Direct compound interaction with the human ether-a-go-go- related gene (hERG) product, a delayed rectifier potassium channel, has been identified as a molecular mechanism of IKr block. One strategy to identify compounds with hERG liability is to monitor hERG current inhibition using electrophysiology techniques. The authors describe the Ion Works HT instrument as a tool for screening cell lines expressing hERG channels. Based on current amplitude and stability criteria, a cell line was selected and used to perform a 300-compound screen. The screen was able to identify compounds with hERG activity within projects that spanned different therapeutic areas. The cell line selection and optimization, as well as the screening abilities of the Ion Works HT system, provide a powerful means of assessing hERGactive compounds early in the drug discovery pipeline.     

Synthesis and biological evaluation of some new 1,4-dihydropyridines containing different ester substitute and diethyl carbamoyl group as anti-tubercular agents

Tuberculosis is a leading infectious cause of death worldwide. Because of the concern of the resistance to most of the commonly used drugs displayed by the considered mycobacteria, most efforts have been done to introduce new anti-tubercular agents. Recent studies showed that 1,4-dihydropyridine-3,5-dicarbamoyl derivatives with lipophilic groups have significant anti-tubercular activity. In this study, we synthesized new derivatives of 1,4-dihydropyridines in which different alkyl and aryl esters and diethyl carbamoyl are substituted in C-3 and C-5 of the DHP ring. In addition nitroimidazole ring is substitutes at C-4 position. These asymmetric analogues were synthesized by a modified Hantzsh reaction using procedure reported by Meyer. The in vitro anti-tubercular activity of compounds against Mycobacterium tuberculosis was evaluated. The results indicate that the compounds containing aromatic esters are more potent than alkyl ones. The most potent aromatic compound (R = 3-phenylpropyl) exhibits comparable anti-tubercular activity (MIC = 1 μmol/ml) with reference compound isoniazide (INH) (MIC = 1 μmol/ml). Conformational analysis, SAR studies of these compounds showed that increasing in lipophilicity and rotable bonds of these compounds resulted in increasing anti-tubercular activity.     

The discovery of novel cyclohexylamide CCR2 antagonists

As a result of further SAR studies on a piperidinyl piperidine scaffold, we report the discovery of compound 44, a potent, orally bioavailable CCR2 antagonist. While having some in vitro hERG activity, this molecule was clean in an in vivo model of QT prolongation. In addition, it showed excellent efficacy when dosed orally in a transgenic murine model of acute inflammation.     

Structure-activity relationships for analogs of the tuberculosis drug bedaquiline with the naphthalene unit replaced by bicyclic heterocycles

Replacing the naphthalene C-unit of the anti-tuberculosis drug bedaquiline with a range of bicyclic heterocycles of widely differing lipophilicity gave analogs with a 4.5-fold range in clogP values. The biological results for these compounds indicate on average a lower clogP limit of about 5.0 in this series for retention of potent inhibitory activity (MIC₉₀s) against M.tb in culture. Some of the compounds also showed a significant reduction in inhibition of hERG channel potassium current compared with bedaquiline, but there was no common structural feature that distinguished these.     

Quinazolin-4-piperidin-4-methyl sulfamide PC-1 inhibitors: Alleviating hERG interactions through structure based design

PC-1 (NPP-1) inhibitors may be useful as therapeutics for the treatment of CDDP (calcium pyrophosphate dehydrate) deposition disease and osteoarthritis. We have identified a series of potent quinazolin-4-piperidin-4-ethyl sulfamide PC-1 inhibitors. The series, however, suffers from high affinity binding to hERG potassium channels, which can cause drug-induced QT prolongation. We used a hERG homology model to identify potential key interactions between our compounds and hERG, and the information gained was used to design and prepare a series of quinazolin-4-piperidin-4-methyl sulfamides that retain PC-1 activity but lack binding affinity for hERG.